Our primary focus is the therapy of hematologic malignancies with recombinant toxins, and in the study of the interaction of toxins with the patient's malignant and normal tissues. Our second, partially overlapping focus is in the development of new treatment paradigms for hairy cell leukemia (HCL), both early and multiply relapsed. 1. Clinical development of anti-CD22 recombinant immunotoxins BL22 and HA22 for HCL. To make a recombinant immunotoxin, the binding domain of a bacterial toxin like Pseudomonas exotoxin (PE) is replaced with a ligand, either a growth factor or an Fv fragment of a monoclonal antibody (MAb), which binds to a tumor associated antigen. The recombinant toxin binds to and is internalized by malignant cells and causes cell death after the catalytic domain of the toxin enters the cytosol of the host cell. BL22 contains an anti-CD22 Fv fused to truncated PE. Data from a phase I and a recently published phase II trial indicate potent and lifesaving activity in chemoresistant hairy cell leukemia. We have reported CR rates of 47-61% overall response rates (ORR) of 72-81%. Disease-free survival (DFS, CR duration) for phase II has not yet been reached at a median of 32 (range 4-62) months, with 12 of 17 CRs still ongoing. Considering all 36 CRs from phase I and II testing, median DFS was 33 (3-112) months with 15 (42%) of 36 CRs ongoing, and two-thirds of the CRs remain in hematologic mission with good blood counts. Smaller spleen size was associated with excellent chance for response (ORR 95%), compared to patients with larger spleens or post-splenectomy, arguing for initiating immunotoxin therapy earlier in the course of disease. The most common serious toxicity was a completely reversible hemolytic uremic syndrome (HUS), and after recovering, patients have maintained normal renal function for a median of 80 months without any evidence of recurrent HUS. Thus, BL22 is highly active producing durable remissions in chemoresistant HCL, particularly in patients with limited disease burden. To improve the targeting of chronic lymphocytic leukemia (CLL), which has lower CD22 density than HCL (median 1250 vs 44,000/cell), BL22 was mutated and a high-affinity recombinant immunotoxin produced, called HA22 or CAT-8015. A phase I trial of HA22 in HCL was recently completed, in which 28 patients were treated. The ORR was 79%, and of the 25 patients in the upper 5 dose levels, the CR rate was 48%. No severe toxicities were observed on this trial and only 2 episodes of grade 2 HUS were observed. Thus HA22 was safe and highly effective in multiply relapsed HCL, and new trials are being planned. Clinical testing in CLL and non-Hodgkins lymphoma is at an earlier stage and will resume when more clinical grade drug is available in the fall of 2009. 2. Clinical development of anti-CD25 recombinant immunotoxin LMB-2 for CD25+ CLL, HCL and cutaneous T-cell lymphoma (CTCL). LMB-2 is markedly effective in CD25+ HCL, particularly in patients who have had HUS with BL22 and cannot receive additional anti-CD22 immunotoxin. Such patients have very limited options and LMB-2 is potentially life-saving for these HCL patients. Activity is also observed as a single agent in CLL and CTCL. Although nearly all patients with CTCL show benefit from LMB-2, retreatment is limited by immunogenicity, possibly due to prior infection with Pseudomonas aeruginosa. Thus LMB-2 may be useful for CTCL patients without prior Pseudomonas exposure. 3. Use of fludarabine and cyclophosphamide (FC) to both prevent immunogenicity and improve response to LMB-2 in patients with adult T-cell leukemia (ATL). To prevent immunogenicity and progression between cycles of LMB-2, patients with ATL, an aggressive T-cell leukemia, are being treated with FC chemotherapy prior to initiating cycles of FC followed by LMB-2. Our hypothesis is that intratumoral (interstitial) soluble CD25 (sCD25) may limit distribution of LMB-2 to rapidly growing tumor cells. So far early clinical results indicate potent clinical activity. Biopsies are also taken to quantify intratumoral sCD25 in the patient tumors. Finally, an animal model is being developed to test the hypothesis using chemotherapy combined with LMB-2 to eradicate CD25+ human tumors. 4. New treatment strategies for early hairy cell leukemia: randomized trial of cladribine + rituximab vs cladribine alone for HCL with either no or only 1 prior therapy. Because evidence of cure for HCL by purine analogs like cladribine is lacking, we have questioned the &gt;20 year standard practice of using cladribine alone as 1st or 2nd line treatment. To determine if the anti-CD20 Mab rituximab can eradicate minimal residual disease (MRD) in HCL and potentially result in cure, we have initiated a randomized trial in which patients receive cladribine combined with rituximab either up front or delayed at least 6 months. This trial is accruing over 2 patients/month, is expected to enroll 150 patients, and should determine whether the early treatment of HCL should be changed. 5. Molecular characterization of HCL with respect to immunoglobulin rearrangements, and assessment of MRD. Discovery and characterization of a new poor-prognosis variant of HCL expressing the VH4-34 immunoglobulin rearrangement, which is also common in autoimmune disorders. By cloning the cDNA for surface IgG displayed on the hairy cells, we have developed a novel sequence-specific PCR test for HCL, able to detect 1 HCL cell in 1,000,000 normal. This is over 10-times more sensitive than flow cytometry and is by far the most sensitive test yet developed for MRD in HCL. We are prospectively cloning surface IgG in patients treated with immunotoxin to determine if BL22 or HA22 can eradicate malignant cells after repeated cycles. We are also constructing a large database of sequences of surface IgG expressed by HCL cells. We find significant differences in expressed IgG between patients with HCL and the poor prognosis variant, HCLv, with respect to both gene usage and mutation frequency. A new variant expressing VH4-34 has been described which has particularly poor response to cladribine alone and poor overall survival from diagnosis, but is sensitive to recombinant immunotoxin. 6. Investigation of the cause of HUS in patients treated with BL22 or HA22. Patients are being prospectively studied to detect markers for this complication of BL22 and HA22. The excretion of BL22, HA22 and LMB-2 is being studied in the urine of patients and animals, and it is found that BL22 is more frequently excreted in the urine than are other agents. Patients are being assayed prospectively for ADAMTS-13 to determine of diminished cleavage of ultralarge multimers of von Willebrands factor is associated with HUS from BL22/HA22. Finally, patients treated with HA22 and BL22 are being prospectively studied with aspirin and/or low molecular weight heparin to determine if these anticoagulants can block immunotoxin-induced HUS. 7. Investigation of tumor markers in patients with B-cell malignancies, including soluble CD22. We found that soluble CD22 (sCD22) could be detected in the serum and its level correlates closely with overall disease burden in both HCL and CLL. sCD25 and sCD22 are being studied in HCL patients after treatment with cladribine, rituximab, or immunotoxins to correlate with other measures of overall disease burden, including flow cytometry, cat scan, and bone marrow biopsy. In a recently published study, a sCD22 level of &lt;2 ng/ml strongly correlated with CR. Our hypothesis i [summary truncated at 7800 characters]